For purposes of this invention, antigen is defined as a reactant from a group consisting of proteins, virus, cells, bacteria, parasites, antibodies and drugs.
Enzyme immunoassay (EIA) is a relatively new analytical technique that has found wide application for the determination of the presence of or concentration of antigenic substances associated with infectious disease, physiological condition, or drug monitoring. EIA techniques have been shown to provide high sensitivity, excellent specificity, and relative simplicity as compared to other immunoassay methods.
Generally, EIA employs the use of an enzyme labeled antibody conjugate directed against the antigen which is being detected or measured, and a primary antibody directed against the antigen. After appropriate incubation and separation procedures, measurement of the enzyme label by colorimetric, spectrophotometric or fluorometric techniques provides a simple and efficient method for detection and quantitation of the antigen.
A difficult and cumbersome problem in EIA methodology has been the separation of the reacted antibody conjugate from the unreacted antibody conjugate. During incubation of the antigen with the primary antibody and enzyme labeled antibody conjugate, a complex is formed by the common attachment of the antigen to the primary antibody and conjugate. Unreacted conjugate, including free and non-specifically bound conjugate must then be separated from the reacted complexed conjugate. After separation and removal of the unreacted conjugate, the antigen-bound enzyme labeled antibody conjugate is measured.
To facilitate the separation of bound from unbound reactants, most EIA techniques in the prior art describe enzyme-linked immunosorbent assays (ELISA). Common to ELISA techniques is the binding or attachment of the primary antibody to a solid-support component or material. The primary antibody is usually attached to a polymeric insoluble solid-support such as a plastic bead. After incubation with the antigen and the enzyme labeled antibody conjugate, the bead is subjected to various washing procedures to remove unreacted conjugate from the system. The bead is then exposed to a substrate for measurement of the bound enzyme conjugate.
A large number of solid-support materials and techniques for use of such materials have been described in the prior art. In one technique, described by Bohn, et al in the U.S. Pat. No. 4,424,279 small beads housed within a plunger device are coated with an antibody to form the solid phase. Deindoerfer, et al in U.S. Pat. No. 3,999,948 describes an antibody-coated solid support consisting of a polymeric film attached to a plastic holder with a handle to facilitate stirring and washing steps. Schall teaches in U.S. Pat. No. 4,363,634 a process for coating a glass support with a polymeric film, with subsequent attachment of an antibody to the film to yield a solid support for immunoassays. Other materials commonly used as solid support components include plastic tubes and microtiter plates, latex and glass particles, and silicone rubber.
The use of cellulose and glass fiber filters for solid support immunoassays has been described. Schutt describes in U.S. Pat. No. 4,357,311 a method for coupling antibody to a filter membrane solid support for use in immunoassay procedures. Katz, et al, in U.S. Pat. No. 4,496,654 describes the bonding of an antibody to an avidin coated paper disc with subsequent use of this paper disc as a solid support in an immunoassay.
Though the use of these solid supports facilitates the separation of reactants in EIA techniques, a number of disadvantages exist. Uniform adsorption of antibody to the solid-support material is required to achieve reproducibility and sensitivity. The ionic charge of the solid support material must be critically controlled to ensure efficient and reproducible adsorption. Numerous problems have been encountered due to lot-to-lot variation in adsorptive characteristics of plastic resins. Antibody adsorbed to these materials is gradually lost from the support surface during the incubation and wash steps, thus reducing the sensitivity of the assay. Non-specific adsorption of the antigen and/or enzyme labeled antibody to the solid-support during incubation may reduce the specificity of the assay.
The use of reactants bound to a solid phase may require long antigen-antibody incubation periods due to inefficient reaction kinetics when one or both reactants are not distributed uniformly throughout the reaction solution. Additionally, steric hindrance may result in less efficient antigen-antibody binding when the reactants are attached to a polymeric support.
Washing procedures employed in ELISA methods are often messy, cumbersome and time-consuming and may be too complicated to be performed in a clinical laboratory. Wash procedures generally involve pouring-off or aspiration of incubation fluids from tubes or microtiter plates followed by addition and removal of copious volumes of wash buffer.
Procedures involving plastic, glass or latex beads may require centrifugation of the incubation fluid followed by removal of supernatants and several additional centrifugation-wash steps. "Dipstick" tests which feature a solid-support material attached to a plastic strip or holder often require moving the test strip sequentially to various tubes of buffer; or, such tests may require rinsing under running tap water for various time intervals.
The present invention overcomes these disadvantages through the use of a novel reactant separation technique which does not require the use of a solid-support or solid phase component.